JP7234598B2 - Nucleic acid amplification reagent and nucleic acid amplification control method using the reagent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a reagent for amplifying a target nucleic acid containing a specific base sequence, which is expected to be contained in a mixture of genes such as DNA and RNA, and a method for controlling nucleic acid amplification using the reagent. Furthermore, the present invention relates to methods and detection reagents for amplifying and qualitatively or quantitatively analyzing said target nucleic acid. INDUSTRIAL APPLICABILITY The present invention is useful for use in the field of clinical diagnosis such as genetic diagnosis, and can amplify and detect target genes directly from specimens. In this case, it is also useful for rapid and highly sensitive identification of the microorganism and detection of drug resistance genes.

In genetic testing used for clinical diagnosis, the amount of nucleic acids in clinical samples is often very small. It realizes accurate measurement.

Examples of the method for amplifying the target nucleic acid include PCR (polymerase chain reaction) method. This method consists of heat denaturation, annealing, and extension reactions using a set of primers consisting of primers complementary and homologous to both ends of a specific DNA sequence in the target nucleic acid, and a thermostable DNA polymerase. By repeating the cycle of steps, a polynucleotide containing the specific DNA sequence can be amplified. On the other hand, when the target nucleic acid is RNA, it is necessary to synthesize cDNA from RNA as a template using reverse transcriptase before performing the PCR method. That is, the procedure is complicated because it requires two steps, a cDNA synthesis step and a PCR reaction step. Moreover, the PCR method requires a special incubator because it requires rapid temperature rise and fall, and is not easy to apply to automation for the purpose of large-scale processing. Furthermore, since it is necessary to repeat the three-step cycle consisting of heat denaturation, annealing, and elongation reaction, there is a limit to speeding up the method.

On the other hand, simple amplification methods for target RNA include NASBA (Nucleic Acid Sequence-Based Amplification) method (Patent Documents 1 and 2, Non-Patent Document 1), TMA (Transcription Mediated Amplification) method (Patent Document 3), TRC (Transcription - Reverse transcription concerted reaction method (Patent Document 4 and Non-Patent Document 2), etc. have been reported. These methods use a primer containing a promoter sequence for a target RNA, reverse transcriptase and ribonuclease H (RNase H) to generate a double-stranded DNA containing a promoter sequence, and a specific base sequence is generated by an RNA polymerase. A chain reaction is then performed using the generated RNA as a template for synthesizing a double-stranded DNA containing the promoter sequence. When amplifying RNA by these methods, an RNA-dependent DNA polymerase activity that synthesizes a complementary DNA strand to the RNA, an RNase H activity that degrades the RNA of the RNA-cDNA duplex, and a DNA that synthesizes a complementary DNA strand to the cDNA. AMV reverse transcriptase, which has a dependent DNA polymerase activity, is commonly used. These amplification methods can be easily applied to automation because the reaction can be performed at a relatively low constant temperature (eg, 41° C. to 46° C.). Furthermore, by adding an enzyme having strand displacement activity, DNA amplification at a relatively low constant temperature is possible (Patent Document 5). However, unlike the PCR reaction, the above-described reaction capable of amplifying the target nucleic acid at a relatively low constant temperature may proceed even at room temperature. Therefore, it has been difficult to start amplification reactions of many specimens at the same time for comparative evaluation.

Real-time PCR is known as a method for quantifying target nucleic acids contained in a sample. However, the efficiency and accuracy of amplification by PCR are influenced by various factors such as the treatment temperature, the base sequence of the nucleic acid, the concentrations and types of reaction solution components, and the like. Therefore, it has been difficult to accurately determine the target nucleic acid amount (concentration) by densitometry of the final PCR product or real-time measurement during the PCR process.

In recent years, digital PCR has been developed as a technique for solving this problem and accurately quantifying minute amounts of target DNA (Non-Patent Document 3). In digital PCR, a solution containing target DNA is distributed to a number of microcompartments and PCR is performed in parallel in each individual microcompartment. Some microcompartments contain one or more target molecules, and some microcompartments do not contain any target molecules. The concentration of target DNA is calculated. As a method for accurately quantifying a small amount of target RNA, cDNA is synthesized in advance using reverse transcriptase, and then the above-mentioned digital PCR is used, or the digital NASBA method (Patent Document 6, and a method using Non-Patent Document 3).

However, since the digital NASBA method uses the NASBA method, which is a reaction to amplify the target nucleic acid at a relatively low constant temperature, as an amplification method, the amplification reaction may proceed even at room temperature. . That is, since the amplification reaction may proceed after preparation of the reaction solution before it is distributed to the microcompartments, highly accurate quantification has been extremely difficult. Therefore, in order to detect an extremely small amount of target RNA present in a sample with high efficiency and good reproducibility, it was necessary to control the amplification reaction, especially at room temperature.

As a control method for target nucleic acid amplification at a constant temperature, magnesium acetate, which is a reaction initiator previously placed in a microcompartment, and a reaction solution are mixed in the microcompartment, followed by RPA (Recombinase Polymerase Amplification). ) method (Patent Document 7) to initiate an isothermal DNA amplification reaction (Non-Patent Document 5). However, the structure of the microfluidic chip is complicated and it is necessary to fabricate a microfluidic chip with a large number of microcompartments. Furthermore, the amplification reaction is dependent on the solubility of magnesium acetate, and in some cases it is conceivable that magnesium acetate aggregates and magnesium does not ionize. As a result, there is a problem that the nucleic acid amplification reaction varies among microcompartments. The occurrence of such errors is fatal in digital nucleic acid amplification.

JP-A-2-005864 Japanese Patent Publication No. 4-503451 Japanese Patent Publication No. 4-500759 Japanese Patent Application Laid-Open No. 2000-014400 JP 2015-116136 A U.S. Publication No. 2014/0141502 Japanese Patent Application Laid-Open No. 2008-188020

de Baar, M.; P. et. al, Journal of Clinical Microbiology, 39, 1895-1902 (2001) Ishiguro, T.; et al, Analytical Biochemistry, 314, 77-86 (2003) Vogelstein, B.; et al, Proc. Natl. Acad. Sci. U.S.A. S. A. , 96, 9236-9241 (1999) Cunningham P.S. R. et al, Biotechniques, 9, 713-714 (1990) S. Yang, A. et al, Science Advances, 3, e1501645 (2017)

The subject of the present invention is a method for amplifying and detecting a very small amount of target nucleic acid present in a sample more efficiently and reproducibly (highly accurately) in a method for amplifying and detecting a target nucleic acid, and the method. To provide a reagent using

The inventors of the present invention have completed the present invention as a result of earnest research to solve the above problems.

That is, aspects of the present invention can be exemplified as follows.

[1] An enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, an enzyme having RNA polymerase activity, and part of a target nucleic acid a first primer having a sequence complementary to the target nucleic acid, a second primer having a sequence homologous to a portion of the target nucleic acid, and a magnesium salt. Either one of the primer and the second primer has a promoter sequence of the enzyme having RNA polymerase activity added to its 5' end),
The amplification reagent further comprising a pyrophosphate degrading enzyme.

[2] The amplification reagent of [1], wherein the magnesium salt is magnesium pyrophosphate.

[3] The amplification reagent of [1] or [2], wherein the enzyme having RNA-dependent DNA polymerase activity, the enzyme having DNA-dependent DNA polymerase activity, and the enzyme having RNase H activity are AMV reverse transcriptases. .

[4] Complementary to a portion of the target nucleic acid located on the 5'-end side of the primer to which the promoter sequence of an enzyme having RNA polymerase activity is added on the 5'-end side (the primer is the first primer any of [1] to [3], further comprising a third primer having a sequence that is identical (if the primer is the second primer) or homologous (if the primer is the second primer), and/or an enzyme that has strand displacement activity amplification reagents.

[5] the amplification reagent according to any one of [1] to [4]; and an oligonucleotide probe whose fluorescence properties change when forming a complementary double strand with a part of the amplified target nucleic acid compared to before formation. A detection reagent for the target nucleic acid, comprising:

[6] A method for amplifying a target nucleic acid, comprising the following steps (1) and (2).
(1) A sample containing a target nucleic acid is treated with an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, and an enzyme having RNA polymerase activity. and a reaction solution containing a first primer having a sequence complementary to a portion of the target nucleic acid, a second primer having a sequence homologous to a portion of the target nucleic acid, and a pyrophosphorolytic enzyme ( However, either one of the first primer and the second primer has a promoter sequence of the enzyme having RNA polymerase activity added to the 5′ end side thereof) (2) sample is added to an initiator containing magnesium pyrophosphate, and nucleic acid amplification reaction is performed at a constant temperature [7] A method for amplifying a target nucleic acid, comprising the following steps (1) to (5).
(1) A sample containing a target nucleic acid is treated with an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, and an enzyme having RNA polymerase activity. and a first primer having a sequence complementary to a portion of the target nucleic acid, and a second primer having a sequence homologous to a portion of the target nucleic acid (provided that the first Either the primer or the second primer has a promoter sequence of the enzyme having RNA polymerase activity added to its 5′ end) (2) the reaction solution to which the sample is added is added to an initiator containing magnesium pyrophosphate or a pyrophosphate degrading enzyme and mixed (3) a step of distributing the mixed solution obtained above into the micropore space (4) magnesium pyrophosphate in the above (2) When adding, a pyrophosphate degrading enzyme is added to the micropore space,
When adding a pyrophosphate degrading enzyme in the above (2), the step of adding magnesium pyrophosphate to the micropore space (5) The step of performing a nucleic acid amplification reaction at a constant temperature [8] In the reaction solution, on the 5' end side Complementary (when said primer is the first primer) or homologous (when said primer is the first primer) or homologous (said primer is the second primer), and/or the amplification method of [6] or [7], further comprising an enzyme having strand displacement activity.

[9] The reaction solution further comprises an oligonucleotide probe whose fluorescence properties change when forming a complementary double strand with a portion of the amplified target nucleic acid compared to before formation, and using the probe, [6] to [ 8] A method for detecting a target nucleic acid, comprising detecting a target nucleic acid amplification product obtained by the amplification method according to any one of [8].

The present invention will be described in detail below.

<1> Amplification Reagent of the Present Invention The amplification reagent of the present invention is a method capable of amplifying single-stranded RNA at a relatively low constant temperature (for example, 41° C. to 46° C.), such as the NASBA method, TMA method, or TRC method. It is characterized by adding a pyrophosphate (PPi) degrading enzyme in addition to the enzymes and primers used. One embodiment of the amplification reagent of the present invention, which is a reagent for amplifying target RNA, is a reagent containing the following components (A) to (J). To either one of (A) and (B) below, a promoter sequence of (G) below is added to the 5'-end side thereof. Other components may also be added that are more preferred for reasons other than those required for commonly used amplification reactions.
(A) a first single-stranded oligonucleotide (first primer) having a sequence complementary to a portion of the target nucleic acid;
(B) a second single-stranded oligonucleotide (second primer) having sequence homology to a portion of the target nucleic acid;
(C) an enzyme having RNA-dependent DNA polymerase activity;
(D) deoxyribonucleoside triphosphates (dNTPs),
(E) an enzyme with RNase H activity;
(F) an enzyme having DNA-dependent DNA polymerase activity;
(G) an enzyme having DNA-dependent RNA polymerase activity;
(H) ribonucleoside triphosphates (NTPs),
(I) PPi-degrading enzyme (J) Magnesium salt The amplified nucleic acid amplified with the amplification reagent of the present invention contains (A) and (B) specific sequence portions to the extent that they can be distinguished from other nucleic acids. can be determined arbitrarily.

As described above, either one of (A) and (B) has the following promoter sequence (G) added to its 5' end. An enhancer sequence consisting of several to several tens of nucleotides may be inserted between the added primer and the promoter sequence.

Of the components (A) to (J), the components (C), (E) and (F) may be different enzymes, or partly or wholly may be a common enzyme. Among them, avian myoblastoma virus (AMV) reverse transcriptase is an enzyme that includes all of the components (C), (E) and (F), and is a particularly preferred embodiment of the amplification reagent of the present invention.

Examples of components of (G) include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. As the promoter sequence to be added to either one of (A) and (B), a sequence corresponding to the component (polymerase) used in (G) may be added.

A feature of the amplification reagent of the present invention is that it contains component (I). The component (I) can decompose an insoluble substance produced by binding PPi, which is a by-product of the nucleic acid amplification reaction, and a component contained in the amplification reagent (for example, the component (J)). Therefore, the amplified product is specifically detected by an optical technique (for example, detection is performed using an oligonucleotide probe whose fluorescence properties change when forming a complementary double strand with a part of the target nucleic acid compared to before formation). In this case, the background derived from the insoluble substance is reduced, and the target nucleic acid contained in the sample can be detected with good reproducibility (high accuracy). Examples of PPi-degrading enzymes include Pyrophosphatase, Inorganic from baker's yeast (S. cerevisiae) [I1643] (manufactured by Sigma-Aldrich), Pyrophosphatase, inorganic (PPase) [10108987001] (manufactured by Roche-Inorganic), Yeast) [M2403], Thermostable Inorganic Pyrophosphatase [M0296], Pyrophosphatase, Inorganic (E. coli) [M0361] (manufactured by New England BioLabs). The amount of component (I) to be added may be appropriately set in consideration of the target nucleic acid to be amplified, the reaction temperature, the amount of PPi that may be generated in the amplification reagent by the nucleic acid amplification reaction of the target nucleic acid, and the like.

The component (J) is an enzyme (components (C), (E), (F) and (G)) necessary for the amplification of the target nucleic acid, and the component (I) has its function (enzymatic activity). is an essential ingredient for Examples of component (J) include water-soluble magnesium salts such as magnesium chloride and magnesium acetate.

It is preferable to use PPi magnesium as the component (J) because the nucleic acid amplification reaction can be controlled by the component (I). When the component (J) is PPimagnesium alone, PPimagnesium itself is a sparingly water-soluble magnesium salt, so the enzyme activity necessary for the amplification of the aforementioned target nucleic acid is almost absent (amplification reaction is suppressed). However, the decomposition reaction of PPi magnesium by magnesium ions generated from slightly dissolved PPi magnesium and the component (I) progresses, and when the magnesium ions contained in the reagent increase, the enzyme necessary for the amplification of the target nucleic acid is inhibited. Activity is restored, allowing amplification of the target nucleic acid. PPi magnesium may be included as one reagent, or may be obtained by adding a water-soluble magnesium salt and PPi as separate reagents and reacting them in a solution. The amount of PPi to be added in the latter may be appropriately set in consideration of the target nucleic acid to be amplified, the reaction temperature, the concentration or other composition of the water-soluble magnesium salt contained in the amplification reagent, and the like.

When the amplification reagent of the present invention containing the components (A) to (J) is used to amplify the target nucleic acid contained in the sample while controlling it, for example, the component (J) is PPi magnesium, and the following The target nucleic acid may be amplified according to the step of .
(a-1) step of adding a sample containing a target nucleic acid to a reaction solution containing components (A) to (I); and performing a nucleic acid amplification reaction at a constant temperature. Note that the operation temperature when adding the initiator containing the component (J) in the step (a-2) is the temperature at which the subsequent nucleic acid amplification reaction is performed. (eg, 41° C. to 46° C.), or a temperature lower than the reaction temperature (eg, ice cooling around 0° C. or room temperature around 25° C.). Especially when the component (I) is a thermostable enzyme (e.g. Thermostable Inorganic Pyrophosphatase [M0296] manufactured by New England BioLabs), the latter temperature allows the control of the target nucleic acid amplification reaction by the components (I) and (J). can be done more efficiently.

When the amplification reagent of the present invention is applied to the amplification of target DNA, a method capable of amplifying single-stranded RNA at a relatively low constant temperature (for example, 41°C to 46°C), such as the NASBA method, TMA method, or TRC method. In addition to the enzymes and primers used in , and the pyrophosphorus degrading enzyme, any one of the following components (K) to (M) may be added. Note that the strand displacement enzyme is an enzyme having strand displacement activity, and in one embodiment of the present invention, when applied to an amplification reaction, it dissociates the already synthesized DNA into single strands, thereby accelerating the amplification reaction. It is preferable to demonstrate Also, the third primer is preferable in one embodiment of the present invention because it exhibits a trigger function to disassociate DNA into single strands.
(K) Enzyme having strand displacement activity (L) Complementary to a portion of the target nucleic acid located on the 5'-end side of the primer having a promoter sequence of an enzyme having RNA polymerase activity added to the 5'-end side Component (K) of component (K) of third primer (M) (K) and (L) having a sequence (when said primer is the first primer) or homologous (when said primer is the second primer) Examples include Bsu DNA polymerase, Bst DNA polymerase (Large Fragment), 96-7 DNA polymerase.

<2> Micro-compartmentalization method When the amplification reagent of the present invention is used to digitally amplify a target nucleic acid, it is necessary to micro-compartmentize the amplification reagent of the present invention. As a method for microcompartmentalization, a special plate with micropores is used to distribute the reaction liquid into the micropores (micropore distribution method), or a special emulsifier or microchannel chip is used to distribute the reaction liquid. There is a method (droplet method) in which the liquid is divided into a large number of minute droplets (droplets). The size of the microcompartments is preferably 5 nL or less, more preferably 2 nL or less, even more preferably 1 nL or less. QuantStudio 3D digital PCR system manufactured by Thermo Fisher Scientific and Clarity digital PCR system manufactured by JN Medsys are commercially available as apparatuses for microcompartmentalization by the micropore distribution method. As devices for microcompartmentalization by the droplet method, the QX200™ Droplet Digital PCR system manufactured by Bio-Rad and the RainDrop Plus Digital PCR system manufactured by RainDance Technologies are commercially available.

<3> Digital Nucleic Acid Amplification Method When performing digital nucleic acid amplification using the microcompartmentalized reagent in <2>, the two liquids may be mixed to initiate the amplification reaction. For example, there is a method in which droplets are generated (microcompartmentalization) by using a microfluidic chip, immediately after forming a two-liquid parallel flow of the dispersed phase through a Y junction, by the fluid resistance force of the sheath flow of the continuous phase. , and using a SlipChip to slip two surfaces facing each other (a first surface having a plurality of first microcompartmental areas and a second surface having a plurality of second microcompartmental areas) to form the By connecting the plurality of first micro-partitioned areas and the plurality of second micro-partitioned areas at a ratio of 1:1, the liquid in each micro-partition can be mixed.

When the micropore distribution method is adopted as the microcompartmentalization method <2>, after mixing the sample containing the target nucleic acid with the solution containing the components (A) to (H) and (J), ( A nucleic acid amplification reaction may be performed by adding the component I) separately, and after mixing the sample containing the target nucleic acid with the solution containing the components (A) to (I), the component (J) is added. The nucleic acid amplification reaction may be performed by adding separately. Specifically, for example, the component (J) is PPi magnesium, and the target nucleic acid is obtained according to the steps shown in (b-1) to (b-4) or (c-1) to (c-4) below. It should be amplified.

(b-1) adding a sample containing a target nucleic acid to a reaction solution containing the components (A) to (H); (b-2) adding the component (J) to the reaction solution to which the sample has been added; (b-3) distributing the solution containing the initiator into the micropore space (b-4) adding the component (I) to the micropore space, and at a constant temperature (c-1) adding a sample containing a target nucleic acid to a reaction solution containing the components (A) to (H); (c-2) adding the sample to the reaction solution; Step (c-3) of adding an initiator containing the component of I) (c-3) Step of distributing the solution containing the initiator into the fine pore space (c-4) Adding the component (J) to the fine pore space Then, the addition of the component (I) in the step of nucleic acid amplification reaction at a constant temperature (b-4) is performed, for example, by adding a solution containing the component (I) to the micropore space in advance, It may be carried out by contacting with the solution distributed in step (b-3). Alternatively, the component (I) may be previously applied or immobilized on the walls of the micropore spaces, and then brought into contact with the solution distributed in the step (b-3). Further, the addition of the component (J) in the step (c-4) is performed by, for example, adding a solution containing the component (J) to the micropore space in advance and then distributing it in the step (c-3). It may be carried out by contacting with a solution containing water. Alternatively, the component (J) may be previously applied or immobilized on the walls of the micropore space, and then brought into contact with the solution distributed in the step (c-3). The immobilization method is not limited as long as the component (I) or the component (J) has activity, and may be physical immobilization or chemical immobilization. Biological immobilization such as antibody reaction or immobilization using biotin-avidin (streptavidin) may also be used.

<4> Detection method A target nucleic acid amplified with the amplification reagent of the present invention can be detected by adding a detection component in advance or after the amplification reaction, and measuring the fluorescence or chemiluminescence intensity derived from the component. good. A preferred embodiment of the component for detection is an oligonucleotide probe whose fluorescent properties change when it forms a complementary double strand with a portion of the amplified target nucleic acid compared to before formation.

An example of the probe is a DNA bound with an intercalating fluorescent dye having a sequence complementary or homologous to a portion of the target nucleic acid. The sequence of the DNA portion should be a sequence that is present in the target nucleic acid and that is complementary or homologous to a portion that is sufficiently distinguishable from nucleic acids other than the target nucleic acid. The length of the DNA portion is preferably 6 to 100 nucleotides, more preferably 10 to 30 nucleotides, for specific analysis of target nucleic acids. In addition, when the DNA part forms a complementary bond with the amplified target nucleic acid, the amplified target nucleic acid is preferably added with a non-complementary sequence or chemically modified (eg, aminated) at its 3' end.

The intercalating fluorescent dye is such that when the aforementioned DNA portion forms a complementary bond with another nucleic acid, it intercalates with the double-stranded portion and changes its fluorescent properties. For this purpose, for example, an intercalating fluorescent dye may be bound to DNA via a linker of suitable molecular length that does not interfere with intercalation into the double-stranded portion. Such a linker is not particularly limited as long as it is a molecule that does not interfere with the intercalation of the intercalating fluorescent dye into the double-stranded portion. In particular, a linker molecule selected from bifunctional hydrocarbons having functional groups at both ends is convenient and preferred for modifying oligonucleotides. A commercially available reagent set (eg, C6-Thiolmodifier manufactured by Clontech) may also be used.

The intercalating fluorescent dye is not particularly limited as long as it changes its fluorescence characteristics, for example, the wavelength of the emitted fluorescence fluctuates by intercalating into the double strands. From such viewpoints, it is particularly preferable to have a property that fluorescence intensity increases by intercalation. Specifically, thiazole orange, oxazole yellow, and derivatives thereof, which exhibit a particularly remarkable change in fluorescence intensity, can be exemplified as preferred intercalating fluorescent dyes.

The position at which the intercalating fluorescent dye is bound to the DNA portion via the linker is such as the 5′ end, 3′ end, or central portion of the DNA portion, which prevents intercalation of the intercalating fluorescent dye into the double strand. There is no particular limitation as long as it does not inhibit complementary binding between the DNA portion and RNA.

Molecular beacons are another example of oligonucleotide probes that change their fluorescent properties when formed into complementary duplexes with a portion of an amplified target nucleic acid compared to before formation. A molecular beacon is a DNA having a sequence complementary or homologous to a portion of a target nucleic acid, and has a stem-loop structure having a fluorescent dye and a quencher at both ends. In the state of the stem-loop structure, the fluorescence of the fluorescent dye is suppressed by the quencher, but when the region complementary to the target RNA present in the loop sequence hybridizes with the amplification product generated during the reaction, the stem portion is cleaved. and emit light. A stem can be formed by forming a double strand of DNA in a molecule and introducing a pair of a fluorescent dye (such as Cy3) and a quencher (such as an azo compound) with high association ability into the DNA.

Yet another example of an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a portion of an amplified target nucleic acid compared to that before formation is a FRET (Fluorescence Resonance Energy Transfer) probe. In this case, two probes designed inside the first and second primers used for amplification are used in the sequence of the target nucleic acid. Of the two probes, the 3' end of one probe is modified with a donor fluorescent dye, and the 5' end of the other probe is modified with an acceptor fluorescent dye. By designing the donor fluorescent dye and the acceptor fluorescent dye to be close to each other when the DNA sequences of the two probes are hybridized, the FRET phenomenon occurs when the two probes hybridize with the amplified product generated during the reaction. detectable.

Yet another example of an oligonucleotide probe that changes its fluorescent properties when formed into a complementary duplex with a portion of an amplified target nucleic acid compared to that before formation is a TaqMan probe. In this case, one probe designed inside the first and second primers used for amplification is used in the sequence of the target nucleic acid. The 5' end of the TaqMan probe is labeled with a fluorescent dye (reporter dye), and the 3' end is labeled with a quencher. The fluorescence of the reporter dye is suppressed by a quencher, but if the probe is hybridized to the target RNA during the extension reaction of the primer by DNA polymerase, the probe is decomposed by 5′→3′ exoclease activity and the reporter dye can be detected by the fluorescence generated by liberation.

The target nucleic acid amplification reagent of the present invention comprises an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H activity, an enzyme having RNA polymerase activity, and a portion of the target nucleic acid that is complementary to a first primer having a similar sequence, a second primer having a sequence homologous to a portion of the target nucleic acid, and an amplification reagent for the target nucleic acid containing a magnesium salt (however, the first primer and the second A promoter sequence of an enzyme having RNA polymerase activity is added to the 5' end of either one of the primers of (1), and further comprises a pyrophosphorolytic enzyme.

According to the present invention, it is possible to control the nucleic acid amplification reaction at room temperature in the amplification reaction of the target nucleic acid contained in the sample at a relatively low constant temperature. Therefore, it is possible to start amplification reactions of target nucleic acids contained in various samples at the same time for comparative evaluation. In addition, since the generation of insoluble substances, which causes an increase in the background, can be suppressed, the target nucleic acid contained in the sample can be amplified and detected with high efficiency and good reproducibility (high precision).

FIG. 2 shows the results of Example 1 and Comparative Example 1 (fluorescence profiles). FIG. 10 is a diagram showing the results of the system containing 5 mM of PPi among the results of Example 2 and Comparative Example 2 (fluorescence profile). FIG. 10 is a diagram showing the results of the system containing 7 mM of PPi among the results of Example 2 and Comparative Example 2 (fluorescence profile).

The present invention will be described in more detail below using Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1 Effect of addition of pyrophosphate degrading enzyme on RNA detection (Part 1)
The influence on RNA detection by further adding pyrophosphate (PPi) degrading enzyme to a system capable of amplifying single-stranded RNA at a constant temperature (NASBA method, TMA method, TRC method, etc.) was evaluated.

(1) The hepatitis C virus standard RNA (hereinafter also simply referred to as standard RNA) was prepared by in vitro transcription from a plasmid into which the gene was inserted. The standard RNA (SEQ ID NO: 1) was diluted with water for injection to 10 ⁵ copies/2 μL, and this was used as an RNA sample.

(2) After preparing a reaction solution having the following composition, the above RNA sample was added to 10 ⁵ copies/12 µL of the reaction solution. The INAF probe (SEQ ID NO: 2), which is a standard RNA detection probe, binds an intercalating fluorescent dye, oxazole yellow, via a linker between the 10th C and the 11th G of the sequence. It is a nucleic acid probe. In addition, the first primer (SEQ ID NO: 3) has a T7 promoter sequence ( It is an oligonucleotide to which SEQ ID NO: 6) is added.

Composition of reaction solution: Final concentration after addition of initiator described later (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36)
0.33 mM each dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, TTP
3.3mM ITP
96.3 mM trehalose 50 nM INAF probe (portion of homologous strand of standard RNA [107th to 123rd of SEQ ID NO: 1]: SEQ ID NO: 2)
3.0 μM first primer (SEQ ID NO: 3)
3.0 μM second primer (portion of homologous strand of standard RNA [1st to 16th of SEQ ID NO: 1]: SEQ ID NO: 4)
12.8U AMV reverse transcriptase (manufactured by Life Science)
166U T7 RNA polymerase PPi degrading enzyme (0.2 U Yeast-derived PPi degrading enzyme [M2403], 0.2 U E. coli-derived PPi degrading enzyme [M0361], 4 U thermostable PPi degrading enzyme [M0296] Either) (manufactured by New England BioLabs)
(3) After incubating the above reaction solution at 46° C. for 3 minutes, 12 μL was added to a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI) in which 8 μL of an initiator having the following composition was dispensed in advance. bottom.

Initiator composition: Concentrations are final after addition of initiator (in 20 μL) 18.4 mM Magnesium chloride 90.0 mM Potassium chloride 0.1% (w/v) Tween 20
9.0% (v/v) DMSO
Water for injection or 5 mM PPi
(4) Subsequently, using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, react at 46 ° C. and measure the fluorescence intensity (excitation wavelength 470 nm, fluorescence wavelength 520 nm) of the reaction solution over time for 30 minutes. bottom.

Comparative example 1
Amplification of standard RNA was attempted in the same manner as in Example 1, except that the PPi-degrading enzyme was omitted from the composition of the reaction solution described in Example 1(2).

With the addition of the initiator as 0 minutes, the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.2. Table 1 shows the results with time as the detection time.

In the system containing no PPi-degrading enzyme in the reaction solution (Comparative Example 1), the presence of PPi in the initiator resulted in no detection of the RNA sample or a considerably delayed detection time (that is, the nucleic acid amplification reaction of the standard RNA was completely or significantly suppressed). On the other hand, in the system containing the PPi-degrading enzyme in the reaction solution (Example 1), the standard RNA could be detected even when the initiator contained 5 mM of PPi (that is, the nucleic acid amplification reaction of the standard RNA was maintained. rice field). Based on this, the system capable of amplifying single-stranded RNA at a constant temperature further contains PPi magnesium generated by the reaction between PPi contained in the starting solution and magnesium chloride, thereby controlling nucleic acid amplification by degrading PPi. I know it can be done with enzymes.

On the other hand, the fluorescence profiles obtained in the measurements of Example 1 and Comparative Example 1 are shown in FIG. When compared with a system that does not contain PPi as an initiator, the system that contains a PPi-degrading enzyme in the reaction solution (Example 1) is more fluorescent than the system that does not contain a PPi-degrading enzyme in the reaction solution (Comparative Example 1). It can be seen that the intensity ratio is high. For this reason, by further including a PPi-degrading enzyme in a system capable of amplifying single-stranded RNA at a constant temperature, the generation of PPi-derived insoluble products that may occur in the nucleic acid amplification reaction is suppressed, and as a result, the insoluble product-derived It can be seen that the fluorescence intensity ratio is improved because the background of is reduced.

Example 2 Effect of addition of pyrophosphate degrading enzyme on RNA detection (Part 2)
In order to further clarify the effects of the presence or absence of the addition of the PPi-degrading enzyme, the PPi-degrading enzyme selected from among the degrading enzymes used in Example 1 was selected from among the degrading enzymes that are resistant to reaction at low temperatures and have high heat resistance at 46 ° C. A PPi-degrading enzyme [M0296] (manufactured by New England BioLabs) was selected, and the effect of the addition of the PPi-degrading enzyme on RNA detection was evaluated.

(1) After preparing a reaction solution having the composition described in Example 1(2), the RNA sample prepared in Example (1) was added at 10 ⁵ copies/12 µL of the reaction solution. However, the PPi-degrading enzyme contained in the reaction solution was 4 U of the aforementioned thermostable PPi-degrading enzyme [M0296] (manufactured by New England BioLabs).

(2) After incubating the above reaction solution at 46° C. for 3 minutes, 12 μL was added to 8 μL of an initiator having the following composition.

Initiator composition: Concentrations are final after addition of initiator (in 20 μL) 18.4 mM Magnesium chloride 90.0 mM Potassium chloride 0.1% (w/v) Tween 20
9.0% (v/v) DMSO
2 mM, 5 mM, 7 mM, 10 mM or 15 mM PPi or water for injection (3) Subsequently, using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, the reaction is performed at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) was measured over time for 30 minutes.

Comparative example 2
Amplification of standard RNA was attempted in the same manner as in Example 2, except that the PPi-degrading enzyme was removed from the reaction solution composition described in Example 1(2).

With the addition of the initiator as 0 minutes, the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.2. Table 2 shows the results with time as the detection time. Among the fluorescence profiles obtained in the measurements of Example 2 and Comparative Example 2, the profiles in systems containing 5 mM and 7 mM of PPi in the initiator are shown in FIGS. 2 and 3, respectively.

In the system containing no PPi-degrading enzyme in the reaction solution (Comparative Example 2), the inclusion of PPi in the initiator delayed the detection time of the RNA sample (that is, the nucleic acid amplification reaction of the standard RNA was suppressed). . Specifically, in the system containing 5 mM of PPi as the initiator, the detection time of the RNA sample was significantly delayed, and the rise of the fluorescence intensity ratio in the fluorescence profile was extremely slow (Fig. 2). It can be seen that the nucleic acid amplification reaction is almost suppressed. In addition, in the system containing 7 mM or more of PPi in the initiator, standard RNA could not be detected at all (that is, the nucleic acid amplification reaction of standard RNA was completely suppressed).

On the other hand, in the system containing the PPi degrading enzyme in the reaction solution (Example 2), the system containing PPi in the initiator but not containing the PPi degrading enzyme in the reaction solution (Comparative Example 2). Compared with , the nucleic acid amplification reaction of standard RNA was accelerated. Specifically, in the system containing 5 mM of PPi in the initiator, the detection time of the RNA sample was significantly faster than in the system containing no PPi-degrading enzyme (Comparative Example 2). Also in the fluorescence profile, the rise of the fluorescence intensity ratio is good (Fig. 2), indicating that the nucleic acid amplification reaction of the standard RNA is greatly accelerated. In addition, the standard RNA could not be detected in the system containing no PPi-degrading enzyme (Comparative Example 2), but could be detected in the system containing 7 mM of PPi as the initiator. Also in the fluorescence profile, the rise of the fluorescence intensity ratio is good (Fig. 3), indicating that the nucleic acid amplification reaction of the standard RNA is maintained.

From the above results, by further including PPi magnesium generated by the reaction between PPi contained in the starting solution and magnesium chloride in a system capable of amplifying single-stranded RNA at a constant temperature, the control of nucleic acid amplification can be controlled by PPi. It turns out that it can be done with a degrading enzyme. Even in a system containing a PPi-degrading enzyme, the detection time of an RNA sample is delayed in the presence of PPi compared to in the absence of PPi. Since the fluorescence profile (FIGS. 2 and 3) is very good, it can be said that there is no problem in the nucleic acid amplification reaction of the standard RNA unless an excessive amount of PPi (10 mM or more in the reaction system of this example) is included. .

Claims (8)

An enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, and RN
an enzyme having A polymerase activity, a first primer having a sequence complementary to a portion of the target nucleic acid, a second primer having a sequence homologous to a portion of the target nucleic acid, and magnesium pyrophosphate an amplification reagent for the target nucleic acid comprising:
Either one of the first primer and the second primer has a promoter sequence of an enzyme having RNA polymerase activity added to its 5′ end,
The amplification reagent further comprising a pyrophosphate degrading enzyme. 2. The amplification reagent of claim 1 , wherein the enzyme having RNA-dependent DNA polymerase activity, the enzyme having DNA-dependent DNA polymerase activity and the enzyme having RNase H activity is AMV reverse transcriptase. Complementary to a portion of the target nucleic acid at the 5'-terminal position of the primer having a promoter sequence for an enzyme having RNA polymerase activity added to the 5'-terminal side (when the primer is the first primer), or 3. The amplification reagent according to claim 1 or 2 , further comprising a third primer having a homologous (when said primer is the second primer) sequence and/or an enzyme having strand displacement activity. 4. The target comprising the amplification reagent according to any one of claims 1 to 3 and an oligonucleotide probe whose fluorescence properties change when forming a complementary double strand with a portion of the amplified target nucleic acid compared to before formation. Nucleic acid detection reagent. A method for amplifying a target nucleic acid, comprising the following steps (1) and (2).
(1) A sample containing a target nucleic acid, an enzyme having RNA-dependent DNA polymerase activity, D
An enzyme with NA-dependent DNA polymerase activity and ribonuclease H (RNase
H) an enzyme having an activity, an enzyme having RNA polymerase activity, a first primer having a sequence complementary to a portion of said target nucleic acid, and a second primer having a sequence homologous to a portion of said target nucleic acid and a pyrophosphate degrading enzyme , wherein the first primer
Either one of the mer and the second primer has the RNA poly
(2) Step of adding the reaction solution to which the promoter sequence of an enzyme having melase activity has been added to the reaction solution added with the sample to an initiator containing magnesium pyrophosphate, and subjecting the reaction solution to nucleic acid amplification reaction at a constant temperature. A method for amplifying a target nucleic acid, comprising the following steps (1) to (5).
(1) A sample containing a target nucleic acid, an enzyme having RNA-dependent DNA polymerase activity, D
An enzyme with NA-dependent DNA polymerase activity and ribonuclease H (RNase
H) an enzyme having an activity, an enzyme having RNA polymerase activity, a first primer having a sequence complementary to a portion of said target nucleic acid, and a second primer having a sequence homologous to a portion of said target nucleic acid and a primer , wherein the first primer and the second primer
Either one of the primers has an enzyme having the above-mentioned RNA polymerase activity on its 5' end side.
Step (2) adding the reaction solution to which the original promoter sequence has been added to the reaction solution to which the sample has been added, adding the reaction solution to an initiator containing magnesium pyrophosphate or a pyrophosphorolytic enzyme, and mixing (3) the above Step (4) of distributing the mixed solution obtained in step (4) in the case of adding magnesium pyrophosphate in the above (2), adding a pyrophosphorolytic enzyme to the micropore space,
When adding a pyrophosphate degrading enzyme in (2) above, step (5) of adding magnesium pyrophosphate to the micropore space and performing a nucleic acid amplification reaction at a constant temperature. Complementary to a portion of the target nucleic acid at the position on the 5'-end side of the reaction solution, to which the promoter sequence of an enzyme having RNA polymerase activity is added on the 5'-end side (the primer is the first primer ) or homologous (when the primer is the second primer) sequence , and/or an enzyme having strand displacement activity . Further comprising in the reaction solution an oligonucleotide probe whose fluorescent properties change when forming a complementary double strand with a part of the amplified target nucleic acid compared to before formation, and using the probe, 7. A method for detecting a target nucleic acid, comprising detecting a target nucleic acid amplification product obtained by the amplification method according to any one of 7 .

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